Study on the anti-endotoxin effect of sinomenine using an Agilent genome array

Study on the anti-endotoxin effect of sinomenine using an Agilent genome array Abstract Background Endotoxin is a significant contributing factor underlying the occurrence of fever, diarrhea, inflammation, edema, coagulation, shock and other syndromes associated with gram-negative bacterial infections. To date, there is no effective treatment for endotoxemia. Aim The aim of this study was to characterize differentially expressed genes in sinomenine-treated and lipopolysaccharide (LPS)-induced endothelial cells by microarray analysis and to determine the potential pharmacological activity of sinomenine. Design The cultured cells of five treatment groups (n = 3) were collected. Participants: total RNA was extracted and subjected to Agilent Porcine 4 × 44 K whole genome microarray. Methods Kyoto encyclopedia of genes and genomes and gene ontology software were applied to screen and analyze differentially regulated genes. Results The results showed that 723 differentially regulated genes were identified including 410 up-regulated genes and 313 down-regulated genes in therapy group vs. LPS group. Ten genes may be key controlled genes in the pathogenesis of LPS, including five up-regulated genes (ARG1, TLR2, IL1A, VCAM1, DKK3) and five down-regulated genes (HABP2, ID1, CHDH, GPX3, PTGFR), which primarily contribute to biological processes such as inflammatory response, vascular lesion, metabolic process and cell cycle. IL1A and FMO3 were considered as potent target genes. Conclusion Global gene expression profile analysis showed that sinomenine might effectively be useful to regulate inflammatory responses as part of future anti-endotoxin therapies. Introduction Lipopolysaccharide (LPS), an endotoxin released by gram-negative bacteria, is the main pathogenic factor of colibacillosis and is largely responsible for morbidity/mortality associated with the disease.1 LPS activates a series of signal transduction pathways in cells and causes pathological effects.2 LPS can injure the microcirculation and vascular endothelial cells by promoting excessive secretion of immune-associated cytokines, thereby contributing to the inflammatory cascade response that ultimately triggers multiple organ dysfunction syndromes.3 Endotoxin is a significant contributing factor underlying the occurrence of fever, diarrhea, inflammation, edema, coagulation, shock and other syndromes associated with gram-negative bacterial infections.4 To date, there is no effective treatment for endotoxemia. Many Chinese herbal medicines can effectively suppress and kill bacterial pathogens. They are widely used to prevent and cure infectious diseases, often with a high efficacy, low toxicity, less occurrence of resistance and lower residual levels remaining in a body than many common drugs. Along these lines, many studies have focused on use of Chinese herbal medicines to mitigate/prevent LPS-induced damage.5 Sinomenine, a pure compound extracted from the Sinomenium acutum plant, has been shown to impart an anti-inflammatory effect in the treatment of immune-related disorders in experimental animal models and in some clinical applications.6 Further investigations using isolated mouse neutrophils confirmed there was indeed an up-regulation of AR induced by sinomenine and that AR-cAMP-PKA signaling was involved in this induced anti-inflammatory effect by this alkaloid.7 Endothelial cells play important roles in a number of physiologic and pathologic processes, such as inflammation, fever, diarrhea, coagulation and shock.8 Regarding initiation/development of immune responses, endothelial cells can be activated by pathogens, leading to their release of various endogenous compounds that modulate vascular relaxation/constriction, including some cytokines. As it has been confirmed that endothelial cells are important primary targets for systemic diseases,9 iliac artery endothelial cells (including those from pigs, i.e. Porcine iliac artery endothelial cells (PIECs)) represent good models for such studies of induced dysfunction. Gene chip technology, with its characteristics of high-throughput simultaneous analysis, automatic rapid analysis and multiparameter sensitive analysis, is useful for the study of gene function and the interactions between genes.10 In this study, changes in the levels of gene expression in PIEC treated with LPS and sinomenine were evaluated using an Agilent Porcine Genome 4 × 44k Array. This array covers 43 603 transcripts and their mutants, representing more than 35 000 functional porcine genes. In this study, the effects of sinomenine as an anti-endotoxin and/or anti-inflammatory agent were evaluated. It was hoped that these studies would provide a theoretical basis for further research on the potential for certain types of alkaloids to be used as novel anti-endotoxin/-inflammatory agents. Materials and methods Reagents Pure sinomenine (at 20 mg/vial; lot #110774) were purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). Sinomenine was diluted to 10 mg/ml with Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) containing 10% fetal bovine serum (FBS; Sigma, St. Louis, MO), 100 U/mL penicillin and 10 μg/ml streptomycin (Gibco, Grand Island, NY). The diluted solutions were then filtered through a 0.22-μm membrane and stored at 4 °C. Before use, the agents were diluted 10-fold with medium. LPS from Escherichia coli (Type O55: B5; Sigma, St. Louis, MO) was prepared following the same procedure as earlier. Experimental protocol PIECs were obtained from the Committee on Type Culture Collection of Chinese Academy of Sciences; the providers indicate that the line was developed from a single host animal. When cultured single-layer endothelial cells reached confluency in a cell incubator (BB15; Thermo Scientific, Chicago, IL), they were allocated into six groups. In blank group, the complete medium (containing 10% FBS) was replaced with maintenance medium (containing 2% FBS) without LPS or sinomenine. In LPS group, the medium was replaced with medium containing LPS at 1 μg/ml. In therapy group, endothelial cells were initially challenged with 1 μg/ml LPS, 3 h later, treated with sinomenine at a concentration of 1 mg/ml for 24 h. In prevention group, endothelial cells were initially challenged with 1 mg/ml sinomenine, 3 h later, treated with LPS at a concentration of 1 μg/ml for 24 h. In meanwhile group, endothelial cells were meanwhile challenged with 1 μg/ml LPS and 1 mg/ml sinomenine for 24 h. In sinomenine group, endothelial cells were challenged with 1 mg/ml sinomenine for 24 h. RNA isolation and cDNA, cRNA synthesis Total RNA was isolated from PIECs in each group according to the manual of the Trizol reagent (Invitrogen Corporation, Carlsbad, California, USA)11 and then purified following the Rneasy protocol.12 The quality of the total RNA samples was assessed by optical density measurement at 260/280 nm and agarose electrophoresis (180 V, 0.5 h) with a 2:1 ratio of 28 S rRNA to 18 S rRNA intensities. Five microgram of total RNA was used as a template for cDNA synthesis. cDNA purification, biotinylated cRNA synthesis and purification were performed by following the manufacturer’s instructions (Agilent).13 The quality and concentration of cDNA and cRNA were examined using a previously reported procedure.14 cRNA fragmentation and microarray detection Fifteen microliter (1 μg/μl) of cRNA was incubated with 5 × fragmentation buffer at 94 °C for 35 min to digest the cRNA into 35–200 bp fragments. The hybridization buffer (prepared according to Agilent protocol) was added to the Agilent Porcine Genome 4 × 44k Array (Agilent technologies Santa Clara, US), and then hybridization was carried out at 45 °C for 16 h on a rotary mixer at 60 rpm. The microarray was washed and stained on a GeneChip fluidics station 450 (Agilent technologies Santa Clara, US) and scanned by GeneChip Scan 3000 (Agilent technologies Santa Clara, US).15 Raw data representing the signal values of gene expression were processed with the GeneChip Operating Software (GCOS) 1.4.16 Statistical analyses The normalized signal values, signal detections and experiment/control were obtained by quantifying and normalizing the signal values, using GCOS 1.4. The probe signal values were scaled to evaluate gene expression (P < 0.05), marginal expression (0.05 < P < 0.065) and no expression (P > 0.065). The signal values of each chip were normalized and evaluated to determine whether a gene’s expression had changed by the ratios, which compared the normalized P values of the treated group to that of the control group, e.g. a gene whose ratio value ≥2 was regarded as up-regulated expression; a gene whose ratio value ≤2 was regarded as down-regulated.17 Statistics and cluster analyses were conducted on these values with GeneMath, GeneSpring (Silicon Genetics, San Carlos, CA, USA) and Microsoft Excel Software (Microsoft, Redmond, WA, USA).18 Results LPS group vs. blank group Eight hundred and fifty-nine genes were up-regulated and 863 genes were down-regulated out of a total of 1722 genes whose expressions showed significant differences in LPS group vs. blank group. Details of the genes whose expressed ratios were obvious are shown in Table 1. Differentially expressed genes mainly involved inflammatory disease, vascular lesion and positive regulation of toll-like receptor signaling pathway. It proved that the pathological model was successful and the effect of endotoxin on cells was extensive. Hierarchical cluster analysis of differentially expressed genes showed that the genes were well-distinguished between the normal and experimental groups (Figure 1). Table 1 Details of some genes whose expressions showed significant differences in LPS group vs. blank group Gene symbol  Fold change  Genbank accession  Gene name  ARG1  315.836  NM_214048  Arginase 1  TLR2  24.455  NM_213761  Toll-like receptor 2  IL1A  15.204  XM_013987857  Interleukin 1, alpha  VCAM1  8.087  NM_213891  Vascular cell adhesion molecule 1  DKK3  5.88  NM_001039749  dickkopf WNT signaling pathway inhibitor 3  HABP2  0.021  NM_001243690  Hyaluronan binding protein 2  ID1  0.079  NM_001244700  Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein  CHDH  0.139  AK393413  Choline dehydrogenase  GPX3  0.213  NM_001115155  Glutathione peroxidase 3  PTGFR  0.387  FJ750950  Prostaglandin F receptor (FP)  Gene symbol  Fold change  Genbank accession  Gene name  ARG1  315.836  NM_214048  Arginase 1  TLR2  24.455  NM_213761  Toll-like receptor 2  IL1A  15.204  XM_013987857  Interleukin 1, alpha  VCAM1  8.087  NM_213891  Vascular cell adhesion molecule 1  DKK3  5.88  NM_001039749  dickkopf WNT signaling pathway inhibitor 3  HABP2  0.021  NM_001243690  Hyaluronan binding protein 2  ID1  0.079  NM_001244700  Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein  CHDH  0.139  AK393413  Choline dehydrogenase  GPX3  0.213  NM_001115155  Glutathione peroxidase 3  PTGFR  0.387  FJ750950  Prostaglandin F receptor (FP)  Figure 1 View largeDownload slide Hierarchical cluster analysis of differentially expressed genes in LPS group vs. blank group. Figure 1 View largeDownload slide Hierarchical cluster analysis of differentially expressed genes in LPS group vs. blank group. Therapy group vs. LPS group Four hundred and ten genes were up-regulated and 313 genes were down-regulated out of a total of 723 genes whose expressions showed significant differences in therapy group vs. LPS group. Details of the genes whose expressed ratios were obvious are shown in Table 2. Differentially expressed genes primarily contributed to response to toxic substance, positive regulation of DNA replication, vitamin digestion and absorption. It suggested that sinomenine, as an anti-endotoxin drug, has a wide range of repair and improvement effects on damaged cells. Volcano plot analysis was performed to represent the differentially expressed mRNA between therapy group vs. LPS group (Figure 2). Table 2 Details of some genes whose expressions showed significant differences in therapy group vs. LPS group Gene symbol  Fold change  Genbank accession  Gene name  FMO3  16.364  AK393242  Flavin containing monooxygenase 3  CYP26A1  10.688  NM_001315792  Cytochrome P450, family 26, subfamily A, polypeptide 1  IFIT3  3.911  NM_001204395  Interferon-induced protein with tetratricopeptide repeats 3  DSCC1  3.568  XM_001926423  DNA replication and sister chromatid cohesion 1  TMPO  2.315  AK397594  thymopoietin  ZBTB32  0.020  AK236945  Zinc finger and BTB domain containing 32  IER3  0.162  AK347702  Immediate early response 3  NFKB2  0.293  AK237079  Nuclear factor of κ light polypeptide gene enhancer in B-cells 2 (p49/p100)  CASP3  0.298  AK230496  Caspase 3, apoptosis-related cysteine peptidase  TLR3  0.441  NM_001097444  Toll-like receptor 3  Gene symbol  Fold change  Genbank accession  Gene name  FMO3  16.364  AK393242  Flavin containing monooxygenase 3  CYP26A1  10.688  NM_001315792  Cytochrome P450, family 26, subfamily A, polypeptide 1  IFIT3  3.911  NM_001204395  Interferon-induced protein with tetratricopeptide repeats 3  DSCC1  3.568  XM_001926423  DNA replication and sister chromatid cohesion 1  TMPO  2.315  AK397594  thymopoietin  ZBTB32  0.020  AK236945  Zinc finger and BTB domain containing 32  IER3  0.162  AK347702  Immediate early response 3  NFKB2  0.293  AK237079  Nuclear factor of κ light polypeptide gene enhancer in B-cells 2 (p49/p100)  CASP3  0.298  AK230496  Caspase 3, apoptosis-related cysteine peptidase  TLR3  0.441  NM_001097444  Toll-like receptor 3  Figure 2 View largeDownload slide Volcano plot analysis of differentially expressed genes in therapy group vs. LPS group. Figure 2 View largeDownload slide Volcano plot analysis of differentially expressed genes in therapy group vs. LPS group. Prevention group vs. LPS group Eight hundred and sixty-two genes were up-regulated and 930 genes were down-regulated out of a total of 1792 genes whose expressions showed significant differences in prevention group vs. LPS group. Details of the genes whose expressed ratios were obvious are shown in Table 3. Differentially expressed genes mainly involved cell adhesion molecule production, negative regulation of extrinsic apoptotic signaling pathway, p53 signaling pathway. As a preventive against LPS, sinomenine is more comprehensive effects than therapy group results. Table 3 Details of some genes whose expressions showed significant differences in prevention group vs. LPS group Gene symbol  Fold change  Genbank accession  Gene name  WIF1  3.879  NM_001315718  WNT inhibitory factor 1  LRIG3  2.908  XM_001927828  Leucine-rich repeats and immunoglobulin-like domains 3  PLAA  2.674  AK390633  Phospholipase A2-activating protein  CALM2  2.559  AK231607  Ccalmodulin 2 (phosphorylase kinase, delta)  AK1  2.439  E01858  Adenylate kinase 1  TLR2  0.051  NM_213761  Toll-like receptor 2  IL18  0.144  NM_213997  Interleukin 18  CASP3  0.204  NM_214131  Caspase 3, apoptosis-related cysteine peptidase  TBXAS1  0.275  NM_214046  Thromboxane A synthase 1 (platelet)  FAS  0.331  NM_213839  Fas cell surface death receptor  Gene symbol  Fold change  Genbank accession  Gene name  WIF1  3.879  NM_001315718  WNT inhibitory factor 1  LRIG3  2.908  XM_001927828  Leucine-rich repeats and immunoglobulin-like domains 3  PLAA  2.674  AK390633  Phospholipase A2-activating protein  CALM2  2.559  AK231607  Ccalmodulin 2 (phosphorylase kinase, delta)  AK1  2.439  E01858  Adenylate kinase 1  TLR2  0.051  NM_213761  Toll-like receptor 2  IL18  0.144  NM_213997  Interleukin 18  CASP3  0.204  NM_214131  Caspase 3, apoptosis-related cysteine peptidase  TBXAS1  0.275  NM_214046  Thromboxane A synthase 1 (platelet)  FAS  0.331  NM_213839  Fas cell surface death receptor  Meanwhile group vs. LPS group Seven hundred and twenty-one genes were up-regulated and 418 genes were down-regulated out of a total of 1139 genes whose expressions showed significant differences in meanwhile group vs. LPS group. Details of the genes whose expressed ratios were obvious are shown in Table 4. Differentially expressed genes mainly referred to regulation of vasoconstriction, negative regulation of response to cytokine stimulus, heterotypic cell-cell adhesion. Compared the results of treatment group and prevention group, the number of differentially expressed genes in meanwhile group was sharply reduced. We guess sinomenine and LPS may have direct chemical neutralization or biological antagonism. Kyoto encyclopedia of genes and genomes (KEGG) analysis was showed between meanwhile group vs. LPS group (Figure 3). Table 4 Details of some genes whose expressions showed significant differences in meanwhile group vs. LPS group Gene symbol  Fold change  Genbank accession  Gene name  UPTI  29.951  NM_213871  Plasmin trypsin inhibitor  SEPP1  8.094  NM_001134823  Selenoprotein P, plasma, 1  NRGN  3.355  AK392047  Neurogranin (protein kinase C substrate, RC3)  GRB7  3.26  XM_013979713  Growth factor receptor-bound protein 7  RAMP1  2.95  NM_214199  Receptor (G protein-coupled) activity modifying protein 1  SIRPA  2.608  AJ544724  Signal-regulatory protein alpha  DRAM1  0.232  NM_001190286  DNA-damage regulated autophagy modulator 1  CASP3  0.384  AK230496  Caspase 3, apoptosis-related cysteine peptidase  TLR3  0.402  NM_001097444  Toll-like receptor 3  IL1RAP  0.42  BI340158  Interleukin 1 receptor accessory protein  Gene symbol  Fold change  Genbank accession  Gene name  UPTI  29.951  NM_213871  Plasmin trypsin inhibitor  SEPP1  8.094  NM_001134823  Selenoprotein P, plasma, 1  NRGN  3.355  AK392047  Neurogranin (protein kinase C substrate, RC3)  GRB7  3.26  XM_013979713  Growth factor receptor-bound protein 7  RAMP1  2.95  NM_214199  Receptor (G protein-coupled) activity modifying protein 1  SIRPA  2.608  AJ544724  Signal-regulatory protein alpha  DRAM1  0.232  NM_001190286  DNA-damage regulated autophagy modulator 1  CASP3  0.384  AK230496  Caspase 3, apoptosis-related cysteine peptidase  TLR3  0.402  NM_001097444  Toll-like receptor 3  IL1RAP  0.42  BI340158  Interleukin 1 receptor accessory protein  Figure 3 View largeDownload slide KEGG analysis of differentially expressed genes in meanwhile group vs. LPS group. Figure 3 View largeDownload slide KEGG analysis of differentially expressed genes in meanwhile group vs. LPS group. Sinomenine group vs. Blank group Three hundred and sixty genes were up-regulated and 317 genes were down-regulated out of a total of 677 genes whose expressions showed significant differences in sinomenine group vs. blank group. Details of the genes whose expressed ratios were obvious are shown in Table 5. Differentially expressed genes mainly dealed with defense response to gram-negative bacterium, positive regulation of wound healing and acute inflammatory response. The results showed that sinomenine had good pharmacological effects such as bactericidal, bacteriostatic, anti-swelling, anti-inflammatory and accelerating healing, no obvious toxicity and side effects. Gene ontology (GO) analysis was performed between sinomenine group vs. blank group (Figure 4). Table 5 Details of some genes whose expressions showed significant differences in sinomenine group vs. blank group Gene symbol  Fold change  Genbank accession  Gene name  ARG1  29.997  NM_214048  Arginase 1  CKM  15.373  NM_001129949  Creatine kinase, muscle  TF  14.487  NM_001244653  Transferrin  S100A8  6.289  NM_001160271  S100 calcium binding protein A8  ABCB1  6.09  NM_001308246  ATP-binding cassette, sub-family B (MDR/TAP), member 1  GHITM  3.125  NM_001244382  Growth hormone inducible transmembrane protein  TNFSF10  0.238  NM_001024696  Tumor necrosis factor (ligand) superfamily, member 10  MADCAM1  0.306  NM_001037998  Mucosal vascular addressin cell adhesion molecule 1  STAT1  0.345  NM_213769  Signal transducer and activator of transcription 1, 91kDa  MAPKBP1  0.366  AK351622  Mitogen-activated protein kinase binding protein 1  Gene symbol  Fold change  Genbank accession  Gene name  ARG1  29.997  NM_214048  Arginase 1  CKM  15.373  NM_001129949  Creatine kinase, muscle  TF  14.487  NM_001244653  Transferrin  S100A8  6.289  NM_001160271  S100 calcium binding protein A8  ABCB1  6.09  NM_001308246  ATP-binding cassette, sub-family B (MDR/TAP), member 1  GHITM  3.125  NM_001244382  Growth hormone inducible transmembrane protein  TNFSF10  0.238  NM_001024696  Tumor necrosis factor (ligand) superfamily, member 10  MADCAM1  0.306  NM_001037998  Mucosal vascular addressin cell adhesion molecule 1  STAT1  0.345  NM_213769  Signal transducer and activator of transcription 1, 91kDa  MAPKBP1  0.366  AK351622  Mitogen-activated protein kinase binding protein 1  Figure 4 View largeDownload slide GO analysis of differentially expressed genes in sinomenine group vs. blank group. Figure 4 View largeDownload slide GO analysis of differentially expressed genes in sinomenine group vs. blank group. Discussion The Agilent chip is the most authoritative chip available and its associated GeneChip technology is considered the professional standard in molecular biology research. We analyzed the 1722 genes whose expressions showed significant differences in LPS group vs. blank group and found that a variety of biochemical processes seemed to play a role in the toxicological effects of LPS. The most important category was inflammatory reaction, which includes TLR2, IL1A, VCAM1 and DKK3. The genes encoding these proteins were all up-regulated. These proteins are all involved in the cascade response that amplifies inflammation and causes fever, edema, diarrhea, coagulation, shock and other clinical symptoms in humans and animals. This inflammation cascade is also mainly responsible for the high mortality of bacterial diseases.19 IL1A also promote apoptosis, which leads to cell death and irreversible structural deformation. TLR2 is responsible for the toll-like receptor signaling pathway, VCAM1 is involved in the MAPK signaling pathway, DKK3 is the main node of the WNT signaling pathway, which had already been shown to be involved in LPS-mediated toxicity.20 The number of up-regulated and down-regulated genes in LPS group vs. blank group was almost the same, suggesting that LPS could induce and inhibit the regulation of physiological responses in the normal body. The pathways suppressed included ribosome, oxidative phosphorylation and various kinds of amino acid metabolism. HABP2 and ID1, down-regulated at the same time, would lead to serious defects in protein synthesis and even threaten life.21 Oxidative phosphorylation genes, such as GPX3 and CHDH, if down-regulated simultaneously, would directly inhibit a number of important catalytic enzyme responses and block body metabolism in the pathological state.22 In addition, the up-regulated gene Arg1 participates in the urea cycle and metabolism of arginine and proline; the up-regulated gene Ampd3 is involved in purine metabolism; and the down-regulated gene Ca3 is responsible for nitrogen metabolism. These genes impact on production, circulation and use of amino acids in the body from both positive and negative aspects and alterations of their expressions would have serious consequences for the infected host.23 There were 723 genes whose expressions showed significant differences in therapy group vs. LPS group. Many of the up-regulated genes that were involved in inflammation, apoptosis, and various signaling pathways in LPS group, were not up-regulated and completely recovered to normal levels in therapy group. The majority of the down-regulated genes, which were involved in ribosomes and oxidative phosphorylation in LPS group vs. blank group recovered to normal levels, however a few of them appeared to be up-regulated in therapy group vs. LPS group. Specifically, the up-regulated genes involved in response to toxic substance in therapy group vs. LPS group included FMO3, CYP26A1 and so on. The up-regulated genes related to immune enhancement were IFIT3, TMPO and DSCC1. The data of LPS group showed that LPS could cause a serial of pathological changes in cell and organism, such as inflammation, apoptosis, defects in protein synthesis, blockage of oxidative phosphorylation, while the data of therapy group revealed that sinomenine could simultaneously reduce inflammatory cytokine expression, suppress the transcription of apoptosis genes, accelerate the synthesis of protein, enhance the energy metabolism of cells, thus play the anti-endotoxin effects. The other up-regulated genes in therapy group vs. LPS group were involved in a wide range of enzyme activity and protein fields, including alanyl aminopeptidase, dopa decarboxylase (aromatic L-amino acid decarboxylase), transglutaminase 2, UDP glucuronosyltransferase 1 family, polypeptide A6, acid phosphatase 5, rhophilin, Rho GTPase binding protein 2, annexin A13, and secreted protein, acidic, cysteine-rich (osteonectin). The upregulation of these genes (related amino acid metabolism, hormone synthesis, immune regulation, cell structure and other physiological processes) suggested that sinomenine has a non-specific but comprehensive protective function for the body. Compared with the 410 up-regulated genes, the 313 down-regulated genes in therapy group vs. LPS group were comparatively few in number, most of which were genes and transcribed loci of unclear function. The difference in the number of up-regulated and down-regulated genes in therapy group vs. LPS group also showed that sinomenine’s multiple pharmacology effects on the body in the pathological state are mainly mediated by the induction of gene expression. The results showed that there were 1792 differentially expressed genes of prevention group vs. LPS group. Among them, there were 862 up-regulated and 930 down-regulated genes. Through the preliminary analysis of GO and KEGG database, the differential genes mainly involved cell adhesion molecule production, negative regulation of extrinsic apoptotic signaling pathway, response to toxic substance, positive regulation of DNA replication, vitamin digestion and absorption. These results showed that sinomenine as a preventive agent against endotoxin, the pharmacological effect is more extensive compared with therapy. It has a clear repair and positive effect for antimicrobial, detumescence and wound healing on pathological damage. Genes whose expressions showed significant differences in meanwhile group vs. LPS group appeared to be representative of the combined simultaneous action of sinomenine and LPS. Only 677 differentially expressed genes in 43 603 pig detection genes/transcripts, and up regulation genes and down regulated genes accounted for about half among them. The differential genes mainly involved regulation of vasoconstriction, negative regulation of response to cytokine stimulus, heterotypic cell-cell adhesion and so on. Sinomenine could intervene endotoxin related signaling pathway, such as TNF, p53 and PPAR.24 Compared with the results of treatment group and prevention group, the number of differentially expressed genes in meanwhile group was sharply reduced. We guess sinomenine and LPS may exist directly neutralization of physicochemical or biological antagonistic effect; the mechanism underlying this effect is unclear. The number of up-regulated genes was almost twice as much that of down-regulated genes, in a total of 1139 genes in sinomenine group vs. blank group. Apart from the fields discussed above, the up-regulated genes were also involved in basic physiological processes including amino acid biosynthesis (ARG1), muscle growth, mineral absorption (TF), calcium pump (S100A8), energy metabolism (ABCB1) and growth hormone (GHITM). The down-regulated genes were involved some biochemical aspects, such as acute inflammatory response (TNFSF10), vascular adhesion (MADCAM1), defense response to gram-negative bacterium (STAT1) and positive regulation of wound healing (MAPKBP1). It was proved that sinomenine was safe without obvious toxicity and side effects. Besides our previous basic research confirms that morphology of endothelial cells with 1 mg/ml sinomenine were the same as in cell control. The OD value of endothelial cells treated with 1 mg/ml sinomenine from the MTT assay and had no statistical differences with that of cell control. The present results suggest that sinomenine might potential have a use as part of a therapeutic regimen to treat toxicities from endotoxin exposure, as demonstrated by animal in vivo experiments25 and cell in vitro experiments26 in our laboratory. Most of this effects could be attributed to its anti-endotoxin and anti-inflammatory effects and subsequent impact on the regulation of cytokine formation/release by a variety of cell types in a host.27 This study revealed crucial information on the pharmacological activity of sinomenine and laid a foundation for subsequent gene validation and functional studies. Furthermore, highly represented genes are target genes, IL1A and FMO3. Clearly, key regulatory genes studies are needed to better clarify whether there will ever be any potential utility of sinomenine in clinical settings. Author contributions Conceived and designed the experiments: Yiyi Hu, Kongwang He. Performed the experiments: Yiyi Hu, Bin Li. Analyzed the data: Yiyi Hu, Libin Wen. Contributed reagents/materials/analysis tools: Yiyi Hu, Kongwang. Wrote the paper: Yiyi Hu. Funding This work was supported by National Natural Science Foundation of China [grant no. 31502112] and Special Fund for Public Welfare Industry of Chinese Ministry of Agriculture [grant no. 201403051]. Acknowledgements The authors greatly appreciate the help we received from all of our colleagues and collaborators in these experiments. Conflict of interest: None declared. References 1 Thorgersen EB, Macagno A, Rossetti C, Mollnes TE. Cyanobacterial LPS antagonist (CyP)-a novel and efficient inhibitor of Escherichia coli LPS-induced cytokine response in the pig. Mol Immunol  2008; 45: 3553– 7. Google Scholar CrossRef Search ADS PubMed  2 Mackman N. Lipopolysaccharide induction of gene expression in human monocytic cells. Immunol Res  2000; 21: 247– 51. http://dx.doi.org/10.1385/IR:21:2-3:247 Google Scholar CrossRef Search ADS PubMed  3 Dauphinee SM, Karsan A. Lipopolysaccharide signaling in endothelial cells. 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Expression patterns of the cell junction-associated genes during rat liver regeneration. J Genetics Genomics  2007; 34: 892– 908. http://dx.doi.org/10.1016/S1673-8527(07)60101-5 Google Scholar CrossRef Search ADS   16 Buettner R, Parhofer KG, Woenckhaus M, Wrede CE, Kunz-Schughart LA, Scholmerich J, et al.   Defining high-fat-diet rat models: metabolic and molecular effects of different fat types. J Mol Endocrinol  2006; 36: 485– 501. Google Scholar CrossRef Search ADS PubMed  17 Takahara Y, Takahashi M, Wagatsuma H, Yokoya F, Zhang QW, Yamaguchi M, et al.   Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis. World J Gastroenterol  2006; 12: 6473– 99. Google Scholar CrossRef Search ADS PubMed  18 Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U.S.A  1998; 95: 14863– 8. Google Scholar CrossRef Search ADS PubMed  19 Suganuma T, Irie K, Fujii E, Yoshioka T, Muraki T. Effect of heat stress on lipopolysaccharide-induced vascular permeability change in mice. J Pharmacol Exper Therap  2002; 303: 656– 63. Google Scholar CrossRef Search ADS   20 Bamford S, Ryley H, Jackson SK. Highly purified lipopolysaccharides from Burkholderia cepacia complex clinical isolates induce inflammatory cytokine responses via TLR4-mediated MAPK signalling pathways and activation of NFkappaB. Cell Microbiol  2007; 9: 532– 43. http://dx.doi.org/10.1111/j.1462-5822.2006.00808.x Google Scholar CrossRef Search ADS PubMed  21 Orellana RA, O'Connor PM, Nguyen HV, Bush JA, Suryawan A, Thivierge MC, et al.   Endotoxemia reduces skeletal muscle protein synthesis in neonates. Am J Physiol Endocrinol Metabol  2002; 283: E909– 16. Google Scholar CrossRef Search ADS   22 Coimbra R, Loomis W, Melbostad H, Tobar M, Porcides RD, Hoyt DB. LPS-stimulated PMN activation and proinflammatory mediator synthesis is downregulated by phosphodiesterase inhibition: role of pentoxifylline. J Trauma  2004; 57: 1157– 63. Google Scholar CrossRef Search ADS PubMed  23 Ohta K, Kikuchi T, Miyahara T, Yoshimura N. DNA microarray analysis of gene expression in iris and ciliary body of rat eyes with endotoxin-induced uveitis. Exp Eye Res  2005; 80: 401– 12. http://dx.doi.org/10.1016/j.exer.2004.10.011 Google Scholar CrossRef Search ADS PubMed  24 Tseng SH, Lee HH, Chen LG, Wu CH, Wang CC. Effects of three purgative decoctions on inflammatory mediators. J Ethnopharmacol  2006; 105: 118– 24. http://dx.doi.org/10.1016/j.jep.2005.10.003 Google Scholar CrossRef Search ADS PubMed  25 Hu Y, Mao A, Yu Z, He K. Anti-endotoxin and anti-inflammatory effects of Chinese herbal medicinal alkaloid ingredients in vivo. Microb Pathogen  2016; 99: 51– 5. http://dx.doi.org/10.1016/j.micpath.2016.08.006 Google Scholar CrossRef Search ADS   26 Hu Y, He K, Zhu H. Chinese herbal medicinal ingredients affect secretion of NO, IL-10, ICAM-1 and IL-2 by endothelial cells. Immunopharmacol Immunotoxicol  2015; 37: 324– 8. http://dx.doi.org/10.3109/08923973.2015.1046991 Google Scholar CrossRef Search ADS PubMed  27 Hu YY, He KW, Guo RL. Six alkaloids inhibit secretion of IL-1alpha, TXB(2), ET-1 and E-selectin in LPS-induced endothelial cells. Immunol Invest  2012; 41: 261– 74. http://dx.doi.org/10.3109/08820139.2011.626826 Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png QJM: An International Journal of Medicine Oxford University Press

Study on the anti-endotoxin effect of sinomenine using an Agilent genome array

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Oxford University Press
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© The Author(s) 2017. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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1460-2725
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10.1093/qjmed/hcx234
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Abstract

Abstract Background Endotoxin is a significant contributing factor underlying the occurrence of fever, diarrhea, inflammation, edema, coagulation, shock and other syndromes associated with gram-negative bacterial infections. To date, there is no effective treatment for endotoxemia. Aim The aim of this study was to characterize differentially expressed genes in sinomenine-treated and lipopolysaccharide (LPS)-induced endothelial cells by microarray analysis and to determine the potential pharmacological activity of sinomenine. Design The cultured cells of five treatment groups (n = 3) were collected. Participants: total RNA was extracted and subjected to Agilent Porcine 4 × 44 K whole genome microarray. Methods Kyoto encyclopedia of genes and genomes and gene ontology software were applied to screen and analyze differentially regulated genes. Results The results showed that 723 differentially regulated genes were identified including 410 up-regulated genes and 313 down-regulated genes in therapy group vs. LPS group. Ten genes may be key controlled genes in the pathogenesis of LPS, including five up-regulated genes (ARG1, TLR2, IL1A, VCAM1, DKK3) and five down-regulated genes (HABP2, ID1, CHDH, GPX3, PTGFR), which primarily contribute to biological processes such as inflammatory response, vascular lesion, metabolic process and cell cycle. IL1A and FMO3 were considered as potent target genes. Conclusion Global gene expression profile analysis showed that sinomenine might effectively be useful to regulate inflammatory responses as part of future anti-endotoxin therapies. Introduction Lipopolysaccharide (LPS), an endotoxin released by gram-negative bacteria, is the main pathogenic factor of colibacillosis and is largely responsible for morbidity/mortality associated with the disease.1 LPS activates a series of signal transduction pathways in cells and causes pathological effects.2 LPS can injure the microcirculation and vascular endothelial cells by promoting excessive secretion of immune-associated cytokines, thereby contributing to the inflammatory cascade response that ultimately triggers multiple organ dysfunction syndromes.3 Endotoxin is a significant contributing factor underlying the occurrence of fever, diarrhea, inflammation, edema, coagulation, shock and other syndromes associated with gram-negative bacterial infections.4 To date, there is no effective treatment for endotoxemia. Many Chinese herbal medicines can effectively suppress and kill bacterial pathogens. They are widely used to prevent and cure infectious diseases, often with a high efficacy, low toxicity, less occurrence of resistance and lower residual levels remaining in a body than many common drugs. Along these lines, many studies have focused on use of Chinese herbal medicines to mitigate/prevent LPS-induced damage.5 Sinomenine, a pure compound extracted from the Sinomenium acutum plant, has been shown to impart an anti-inflammatory effect in the treatment of immune-related disorders in experimental animal models and in some clinical applications.6 Further investigations using isolated mouse neutrophils confirmed there was indeed an up-regulation of AR induced by sinomenine and that AR-cAMP-PKA signaling was involved in this induced anti-inflammatory effect by this alkaloid.7 Endothelial cells play important roles in a number of physiologic and pathologic processes, such as inflammation, fever, diarrhea, coagulation and shock.8 Regarding initiation/development of immune responses, endothelial cells can be activated by pathogens, leading to their release of various endogenous compounds that modulate vascular relaxation/constriction, including some cytokines. As it has been confirmed that endothelial cells are important primary targets for systemic diseases,9 iliac artery endothelial cells (including those from pigs, i.e. Porcine iliac artery endothelial cells (PIECs)) represent good models for such studies of induced dysfunction. Gene chip technology, with its characteristics of high-throughput simultaneous analysis, automatic rapid analysis and multiparameter sensitive analysis, is useful for the study of gene function and the interactions between genes.10 In this study, changes in the levels of gene expression in PIEC treated with LPS and sinomenine were evaluated using an Agilent Porcine Genome 4 × 44k Array. This array covers 43 603 transcripts and their mutants, representing more than 35 000 functional porcine genes. In this study, the effects of sinomenine as an anti-endotoxin and/or anti-inflammatory agent were evaluated. It was hoped that these studies would provide a theoretical basis for further research on the potential for certain types of alkaloids to be used as novel anti-endotoxin/-inflammatory agents. Materials and methods Reagents Pure sinomenine (at 20 mg/vial; lot #110774) were purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). Sinomenine was diluted to 10 mg/ml with Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) containing 10% fetal bovine serum (FBS; Sigma, St. Louis, MO), 100 U/mL penicillin and 10 μg/ml streptomycin (Gibco, Grand Island, NY). The diluted solutions were then filtered through a 0.22-μm membrane and stored at 4 °C. Before use, the agents were diluted 10-fold with medium. LPS from Escherichia coli (Type O55: B5; Sigma, St. Louis, MO) was prepared following the same procedure as earlier. Experimental protocol PIECs were obtained from the Committee on Type Culture Collection of Chinese Academy of Sciences; the providers indicate that the line was developed from a single host animal. When cultured single-layer endothelial cells reached confluency in a cell incubator (BB15; Thermo Scientific, Chicago, IL), they were allocated into six groups. In blank group, the complete medium (containing 10% FBS) was replaced with maintenance medium (containing 2% FBS) without LPS or sinomenine. In LPS group, the medium was replaced with medium containing LPS at 1 μg/ml. In therapy group, endothelial cells were initially challenged with 1 μg/ml LPS, 3 h later, treated with sinomenine at a concentration of 1 mg/ml for 24 h. In prevention group, endothelial cells were initially challenged with 1 mg/ml sinomenine, 3 h later, treated with LPS at a concentration of 1 μg/ml for 24 h. In meanwhile group, endothelial cells were meanwhile challenged with 1 μg/ml LPS and 1 mg/ml sinomenine for 24 h. In sinomenine group, endothelial cells were challenged with 1 mg/ml sinomenine for 24 h. RNA isolation and cDNA, cRNA synthesis Total RNA was isolated from PIECs in each group according to the manual of the Trizol reagent (Invitrogen Corporation, Carlsbad, California, USA)11 and then purified following the Rneasy protocol.12 The quality of the total RNA samples was assessed by optical density measurement at 260/280 nm and agarose electrophoresis (180 V, 0.5 h) with a 2:1 ratio of 28 S rRNA to 18 S rRNA intensities. Five microgram of total RNA was used as a template for cDNA synthesis. cDNA purification, biotinylated cRNA synthesis and purification were performed by following the manufacturer’s instructions (Agilent).13 The quality and concentration of cDNA and cRNA were examined using a previously reported procedure.14 cRNA fragmentation and microarray detection Fifteen microliter (1 μg/μl) of cRNA was incubated with 5 × fragmentation buffer at 94 °C for 35 min to digest the cRNA into 35–200 bp fragments. The hybridization buffer (prepared according to Agilent protocol) was added to the Agilent Porcine Genome 4 × 44k Array (Agilent technologies Santa Clara, US), and then hybridization was carried out at 45 °C for 16 h on a rotary mixer at 60 rpm. The microarray was washed and stained on a GeneChip fluidics station 450 (Agilent technologies Santa Clara, US) and scanned by GeneChip Scan 3000 (Agilent technologies Santa Clara, US).15 Raw data representing the signal values of gene expression were processed with the GeneChip Operating Software (GCOS) 1.4.16 Statistical analyses The normalized signal values, signal detections and experiment/control were obtained by quantifying and normalizing the signal values, using GCOS 1.4. The probe signal values were scaled to evaluate gene expression (P < 0.05), marginal expression (0.05 < P < 0.065) and no expression (P > 0.065). The signal values of each chip were normalized and evaluated to determine whether a gene’s expression had changed by the ratios, which compared the normalized P values of the treated group to that of the control group, e.g. a gene whose ratio value ≥2 was regarded as up-regulated expression; a gene whose ratio value ≤2 was regarded as down-regulated.17 Statistics and cluster analyses were conducted on these values with GeneMath, GeneSpring (Silicon Genetics, San Carlos, CA, USA) and Microsoft Excel Software (Microsoft, Redmond, WA, USA).18 Results LPS group vs. blank group Eight hundred and fifty-nine genes were up-regulated and 863 genes were down-regulated out of a total of 1722 genes whose expressions showed significant differences in LPS group vs. blank group. Details of the genes whose expressed ratios were obvious are shown in Table 1. Differentially expressed genes mainly involved inflammatory disease, vascular lesion and positive regulation of toll-like receptor signaling pathway. It proved that the pathological model was successful and the effect of endotoxin on cells was extensive. Hierarchical cluster analysis of differentially expressed genes showed that the genes were well-distinguished between the normal and experimental groups (Figure 1). Table 1 Details of some genes whose expressions showed significant differences in LPS group vs. blank group Gene symbol  Fold change  Genbank accession  Gene name  ARG1  315.836  NM_214048  Arginase 1  TLR2  24.455  NM_213761  Toll-like receptor 2  IL1A  15.204  XM_013987857  Interleukin 1, alpha  VCAM1  8.087  NM_213891  Vascular cell adhesion molecule 1  DKK3  5.88  NM_001039749  dickkopf WNT signaling pathway inhibitor 3  HABP2  0.021  NM_001243690  Hyaluronan binding protein 2  ID1  0.079  NM_001244700  Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein  CHDH  0.139  AK393413  Choline dehydrogenase  GPX3  0.213  NM_001115155  Glutathione peroxidase 3  PTGFR  0.387  FJ750950  Prostaglandin F receptor (FP)  Gene symbol  Fold change  Genbank accession  Gene name  ARG1  315.836  NM_214048  Arginase 1  TLR2  24.455  NM_213761  Toll-like receptor 2  IL1A  15.204  XM_013987857  Interleukin 1, alpha  VCAM1  8.087  NM_213891  Vascular cell adhesion molecule 1  DKK3  5.88  NM_001039749  dickkopf WNT signaling pathway inhibitor 3  HABP2  0.021  NM_001243690  Hyaluronan binding protein 2  ID1  0.079  NM_001244700  Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein  CHDH  0.139  AK393413  Choline dehydrogenase  GPX3  0.213  NM_001115155  Glutathione peroxidase 3  PTGFR  0.387  FJ750950  Prostaglandin F receptor (FP)  Figure 1 View largeDownload slide Hierarchical cluster analysis of differentially expressed genes in LPS group vs. blank group. Figure 1 View largeDownload slide Hierarchical cluster analysis of differentially expressed genes in LPS group vs. blank group. Therapy group vs. LPS group Four hundred and ten genes were up-regulated and 313 genes were down-regulated out of a total of 723 genes whose expressions showed significant differences in therapy group vs. LPS group. Details of the genes whose expressed ratios were obvious are shown in Table 2. Differentially expressed genes primarily contributed to response to toxic substance, positive regulation of DNA replication, vitamin digestion and absorption. It suggested that sinomenine, as an anti-endotoxin drug, has a wide range of repair and improvement effects on damaged cells. Volcano plot analysis was performed to represent the differentially expressed mRNA between therapy group vs. LPS group (Figure 2). Table 2 Details of some genes whose expressions showed significant differences in therapy group vs. LPS group Gene symbol  Fold change  Genbank accession  Gene name  FMO3  16.364  AK393242  Flavin containing monooxygenase 3  CYP26A1  10.688  NM_001315792  Cytochrome P450, family 26, subfamily A, polypeptide 1  IFIT3  3.911  NM_001204395  Interferon-induced protein with tetratricopeptide repeats 3  DSCC1  3.568  XM_001926423  DNA replication and sister chromatid cohesion 1  TMPO  2.315  AK397594  thymopoietin  ZBTB32  0.020  AK236945  Zinc finger and BTB domain containing 32  IER3  0.162  AK347702  Immediate early response 3  NFKB2  0.293  AK237079  Nuclear factor of κ light polypeptide gene enhancer in B-cells 2 (p49/p100)  CASP3  0.298  AK230496  Caspase 3, apoptosis-related cysteine peptidase  TLR3  0.441  NM_001097444  Toll-like receptor 3  Gene symbol  Fold change  Genbank accession  Gene name  FMO3  16.364  AK393242  Flavin containing monooxygenase 3  CYP26A1  10.688  NM_001315792  Cytochrome P450, family 26, subfamily A, polypeptide 1  IFIT3  3.911  NM_001204395  Interferon-induced protein with tetratricopeptide repeats 3  DSCC1  3.568  XM_001926423  DNA replication and sister chromatid cohesion 1  TMPO  2.315  AK397594  thymopoietin  ZBTB32  0.020  AK236945  Zinc finger and BTB domain containing 32  IER3  0.162  AK347702  Immediate early response 3  NFKB2  0.293  AK237079  Nuclear factor of κ light polypeptide gene enhancer in B-cells 2 (p49/p100)  CASP3  0.298  AK230496  Caspase 3, apoptosis-related cysteine peptidase  TLR3  0.441  NM_001097444  Toll-like receptor 3  Figure 2 View largeDownload slide Volcano plot analysis of differentially expressed genes in therapy group vs. LPS group. Figure 2 View largeDownload slide Volcano plot analysis of differentially expressed genes in therapy group vs. LPS group. Prevention group vs. LPS group Eight hundred and sixty-two genes were up-regulated and 930 genes were down-regulated out of a total of 1792 genes whose expressions showed significant differences in prevention group vs. LPS group. Details of the genes whose expressed ratios were obvious are shown in Table 3. Differentially expressed genes mainly involved cell adhesion molecule production, negative regulation of extrinsic apoptotic signaling pathway, p53 signaling pathway. As a preventive against LPS, sinomenine is more comprehensive effects than therapy group results. Table 3 Details of some genes whose expressions showed significant differences in prevention group vs. LPS group Gene symbol  Fold change  Genbank accession  Gene name  WIF1  3.879  NM_001315718  WNT inhibitory factor 1  LRIG3  2.908  XM_001927828  Leucine-rich repeats and immunoglobulin-like domains 3  PLAA  2.674  AK390633  Phospholipase A2-activating protein  CALM2  2.559  AK231607  Ccalmodulin 2 (phosphorylase kinase, delta)  AK1  2.439  E01858  Adenylate kinase 1  TLR2  0.051  NM_213761  Toll-like receptor 2  IL18  0.144  NM_213997  Interleukin 18  CASP3  0.204  NM_214131  Caspase 3, apoptosis-related cysteine peptidase  TBXAS1  0.275  NM_214046  Thromboxane A synthase 1 (platelet)  FAS  0.331  NM_213839  Fas cell surface death receptor  Gene symbol  Fold change  Genbank accession  Gene name  WIF1  3.879  NM_001315718  WNT inhibitory factor 1  LRIG3  2.908  XM_001927828  Leucine-rich repeats and immunoglobulin-like domains 3  PLAA  2.674  AK390633  Phospholipase A2-activating protein  CALM2  2.559  AK231607  Ccalmodulin 2 (phosphorylase kinase, delta)  AK1  2.439  E01858  Adenylate kinase 1  TLR2  0.051  NM_213761  Toll-like receptor 2  IL18  0.144  NM_213997  Interleukin 18  CASP3  0.204  NM_214131  Caspase 3, apoptosis-related cysteine peptidase  TBXAS1  0.275  NM_214046  Thromboxane A synthase 1 (platelet)  FAS  0.331  NM_213839  Fas cell surface death receptor  Meanwhile group vs. LPS group Seven hundred and twenty-one genes were up-regulated and 418 genes were down-regulated out of a total of 1139 genes whose expressions showed significant differences in meanwhile group vs. LPS group. Details of the genes whose expressed ratios were obvious are shown in Table 4. Differentially expressed genes mainly referred to regulation of vasoconstriction, negative regulation of response to cytokine stimulus, heterotypic cell-cell adhesion. Compared the results of treatment group and prevention group, the number of differentially expressed genes in meanwhile group was sharply reduced. We guess sinomenine and LPS may have direct chemical neutralization or biological antagonism. Kyoto encyclopedia of genes and genomes (KEGG) analysis was showed between meanwhile group vs. LPS group (Figure 3). Table 4 Details of some genes whose expressions showed significant differences in meanwhile group vs. LPS group Gene symbol  Fold change  Genbank accession  Gene name  UPTI  29.951  NM_213871  Plasmin trypsin inhibitor  SEPP1  8.094  NM_001134823  Selenoprotein P, plasma, 1  NRGN  3.355  AK392047  Neurogranin (protein kinase C substrate, RC3)  GRB7  3.26  XM_013979713  Growth factor receptor-bound protein 7  RAMP1  2.95  NM_214199  Receptor (G protein-coupled) activity modifying protein 1  SIRPA  2.608  AJ544724  Signal-regulatory protein alpha  DRAM1  0.232  NM_001190286  DNA-damage regulated autophagy modulator 1  CASP3  0.384  AK230496  Caspase 3, apoptosis-related cysteine peptidase  TLR3  0.402  NM_001097444  Toll-like receptor 3  IL1RAP  0.42  BI340158  Interleukin 1 receptor accessory protein  Gene symbol  Fold change  Genbank accession  Gene name  UPTI  29.951  NM_213871  Plasmin trypsin inhibitor  SEPP1  8.094  NM_001134823  Selenoprotein P, plasma, 1  NRGN  3.355  AK392047  Neurogranin (protein kinase C substrate, RC3)  GRB7  3.26  XM_013979713  Growth factor receptor-bound protein 7  RAMP1  2.95  NM_214199  Receptor (G protein-coupled) activity modifying protein 1  SIRPA  2.608  AJ544724  Signal-regulatory protein alpha  DRAM1  0.232  NM_001190286  DNA-damage regulated autophagy modulator 1  CASP3  0.384  AK230496  Caspase 3, apoptosis-related cysteine peptidase  TLR3  0.402  NM_001097444  Toll-like receptor 3  IL1RAP  0.42  BI340158  Interleukin 1 receptor accessory protein  Figure 3 View largeDownload slide KEGG analysis of differentially expressed genes in meanwhile group vs. LPS group. Figure 3 View largeDownload slide KEGG analysis of differentially expressed genes in meanwhile group vs. LPS group. Sinomenine group vs. Blank group Three hundred and sixty genes were up-regulated and 317 genes were down-regulated out of a total of 677 genes whose expressions showed significant differences in sinomenine group vs. blank group. Details of the genes whose expressed ratios were obvious are shown in Table 5. Differentially expressed genes mainly dealed with defense response to gram-negative bacterium, positive regulation of wound healing and acute inflammatory response. The results showed that sinomenine had good pharmacological effects such as bactericidal, bacteriostatic, anti-swelling, anti-inflammatory and accelerating healing, no obvious toxicity and side effects. Gene ontology (GO) analysis was performed between sinomenine group vs. blank group (Figure 4). Table 5 Details of some genes whose expressions showed significant differences in sinomenine group vs. blank group Gene symbol  Fold change  Genbank accession  Gene name  ARG1  29.997  NM_214048  Arginase 1  CKM  15.373  NM_001129949  Creatine kinase, muscle  TF  14.487  NM_001244653  Transferrin  S100A8  6.289  NM_001160271  S100 calcium binding protein A8  ABCB1  6.09  NM_001308246  ATP-binding cassette, sub-family B (MDR/TAP), member 1  GHITM  3.125  NM_001244382  Growth hormone inducible transmembrane protein  TNFSF10  0.238  NM_001024696  Tumor necrosis factor (ligand) superfamily, member 10  MADCAM1  0.306  NM_001037998  Mucosal vascular addressin cell adhesion molecule 1  STAT1  0.345  NM_213769  Signal transducer and activator of transcription 1, 91kDa  MAPKBP1  0.366  AK351622  Mitogen-activated protein kinase binding protein 1  Gene symbol  Fold change  Genbank accession  Gene name  ARG1  29.997  NM_214048  Arginase 1  CKM  15.373  NM_001129949  Creatine kinase, muscle  TF  14.487  NM_001244653  Transferrin  S100A8  6.289  NM_001160271  S100 calcium binding protein A8  ABCB1  6.09  NM_001308246  ATP-binding cassette, sub-family B (MDR/TAP), member 1  GHITM  3.125  NM_001244382  Growth hormone inducible transmembrane protein  TNFSF10  0.238  NM_001024696  Tumor necrosis factor (ligand) superfamily, member 10  MADCAM1  0.306  NM_001037998  Mucosal vascular addressin cell adhesion molecule 1  STAT1  0.345  NM_213769  Signal transducer and activator of transcription 1, 91kDa  MAPKBP1  0.366  AK351622  Mitogen-activated protein kinase binding protein 1  Figure 4 View largeDownload slide GO analysis of differentially expressed genes in sinomenine group vs. blank group. Figure 4 View largeDownload slide GO analysis of differentially expressed genes in sinomenine group vs. blank group. Discussion The Agilent chip is the most authoritative chip available and its associated GeneChip technology is considered the professional standard in molecular biology research. We analyzed the 1722 genes whose expressions showed significant differences in LPS group vs. blank group and found that a variety of biochemical processes seemed to play a role in the toxicological effects of LPS. The most important category was inflammatory reaction, which includes TLR2, IL1A, VCAM1 and DKK3. The genes encoding these proteins were all up-regulated. These proteins are all involved in the cascade response that amplifies inflammation and causes fever, edema, diarrhea, coagulation, shock and other clinical symptoms in humans and animals. This inflammation cascade is also mainly responsible for the high mortality of bacterial diseases.19 IL1A also promote apoptosis, which leads to cell death and irreversible structural deformation. TLR2 is responsible for the toll-like receptor signaling pathway, VCAM1 is involved in the MAPK signaling pathway, DKK3 is the main node of the WNT signaling pathway, which had already been shown to be involved in LPS-mediated toxicity.20 The number of up-regulated and down-regulated genes in LPS group vs. blank group was almost the same, suggesting that LPS could induce and inhibit the regulation of physiological responses in the normal body. The pathways suppressed included ribosome, oxidative phosphorylation and various kinds of amino acid metabolism. HABP2 and ID1, down-regulated at the same time, would lead to serious defects in protein synthesis and even threaten life.21 Oxidative phosphorylation genes, such as GPX3 and CHDH, if down-regulated simultaneously, would directly inhibit a number of important catalytic enzyme responses and block body metabolism in the pathological state.22 In addition, the up-regulated gene Arg1 participates in the urea cycle and metabolism of arginine and proline; the up-regulated gene Ampd3 is involved in purine metabolism; and the down-regulated gene Ca3 is responsible for nitrogen metabolism. These genes impact on production, circulation and use of amino acids in the body from both positive and negative aspects and alterations of their expressions would have serious consequences for the infected host.23 There were 723 genes whose expressions showed significant differences in therapy group vs. LPS group. Many of the up-regulated genes that were involved in inflammation, apoptosis, and various signaling pathways in LPS group, were not up-regulated and completely recovered to normal levels in therapy group. The majority of the down-regulated genes, which were involved in ribosomes and oxidative phosphorylation in LPS group vs. blank group recovered to normal levels, however a few of them appeared to be up-regulated in therapy group vs. LPS group. Specifically, the up-regulated genes involved in response to toxic substance in therapy group vs. LPS group included FMO3, CYP26A1 and so on. The up-regulated genes related to immune enhancement were IFIT3, TMPO and DSCC1. The data of LPS group showed that LPS could cause a serial of pathological changes in cell and organism, such as inflammation, apoptosis, defects in protein synthesis, blockage of oxidative phosphorylation, while the data of therapy group revealed that sinomenine could simultaneously reduce inflammatory cytokine expression, suppress the transcription of apoptosis genes, accelerate the synthesis of protein, enhance the energy metabolism of cells, thus play the anti-endotoxin effects. The other up-regulated genes in therapy group vs. LPS group were involved in a wide range of enzyme activity and protein fields, including alanyl aminopeptidase, dopa decarboxylase (aromatic L-amino acid decarboxylase), transglutaminase 2, UDP glucuronosyltransferase 1 family, polypeptide A6, acid phosphatase 5, rhophilin, Rho GTPase binding protein 2, annexin A13, and secreted protein, acidic, cysteine-rich (osteonectin). The upregulation of these genes (related amino acid metabolism, hormone synthesis, immune regulation, cell structure and other physiological processes) suggested that sinomenine has a non-specific but comprehensive protective function for the body. Compared with the 410 up-regulated genes, the 313 down-regulated genes in therapy group vs. LPS group were comparatively few in number, most of which were genes and transcribed loci of unclear function. The difference in the number of up-regulated and down-regulated genes in therapy group vs. LPS group also showed that sinomenine’s multiple pharmacology effects on the body in the pathological state are mainly mediated by the induction of gene expression. The results showed that there were 1792 differentially expressed genes of prevention group vs. LPS group. Among them, there were 862 up-regulated and 930 down-regulated genes. Through the preliminary analysis of GO and KEGG database, the differential genes mainly involved cell adhesion molecule production, negative regulation of extrinsic apoptotic signaling pathway, response to toxic substance, positive regulation of DNA replication, vitamin digestion and absorption. These results showed that sinomenine as a preventive agent against endotoxin, the pharmacological effect is more extensive compared with therapy. It has a clear repair and positive effect for antimicrobial, detumescence and wound healing on pathological damage. Genes whose expressions showed significant differences in meanwhile group vs. LPS group appeared to be representative of the combined simultaneous action of sinomenine and LPS. Only 677 differentially expressed genes in 43 603 pig detection genes/transcripts, and up regulation genes and down regulated genes accounted for about half among them. The differential genes mainly involved regulation of vasoconstriction, negative regulation of response to cytokine stimulus, heterotypic cell-cell adhesion and so on. Sinomenine could intervene endotoxin related signaling pathway, such as TNF, p53 and PPAR.24 Compared with the results of treatment group and prevention group, the number of differentially expressed genes in meanwhile group was sharply reduced. We guess sinomenine and LPS may exist directly neutralization of physicochemical or biological antagonistic effect; the mechanism underlying this effect is unclear. The number of up-regulated genes was almost twice as much that of down-regulated genes, in a total of 1139 genes in sinomenine group vs. blank group. Apart from the fields discussed above, the up-regulated genes were also involved in basic physiological processes including amino acid biosynthesis (ARG1), muscle growth, mineral absorption (TF), calcium pump (S100A8), energy metabolism (ABCB1) and growth hormone (GHITM). The down-regulated genes were involved some biochemical aspects, such as acute inflammatory response (TNFSF10), vascular adhesion (MADCAM1), defense response to gram-negative bacterium (STAT1) and positive regulation of wound healing (MAPKBP1). It was proved that sinomenine was safe without obvious toxicity and side effects. Besides our previous basic research confirms that morphology of endothelial cells with 1 mg/ml sinomenine were the same as in cell control. The OD value of endothelial cells treated with 1 mg/ml sinomenine from the MTT assay and had no statistical differences with that of cell control. The present results suggest that sinomenine might potential have a use as part of a therapeutic regimen to treat toxicities from endotoxin exposure, as demonstrated by animal in vivo experiments25 and cell in vitro experiments26 in our laboratory. Most of this effects could be attributed to its anti-endotoxin and anti-inflammatory effects and subsequent impact on the regulation of cytokine formation/release by a variety of cell types in a host.27 This study revealed crucial information on the pharmacological activity of sinomenine and laid a foundation for subsequent gene validation and functional studies. Furthermore, highly represented genes are target genes, IL1A and FMO3. Clearly, key regulatory genes studies are needed to better clarify whether there will ever be any potential utility of sinomenine in clinical settings. Author contributions Conceived and designed the experiments: Yiyi Hu, Kongwang He. Performed the experiments: Yiyi Hu, Bin Li. Analyzed the data: Yiyi Hu, Libin Wen. Contributed reagents/materials/analysis tools: Yiyi Hu, Kongwang. Wrote the paper: Yiyi Hu. Funding This work was supported by National Natural Science Foundation of China [grant no. 31502112] and Special Fund for Public Welfare Industry of Chinese Ministry of Agriculture [grant no. 201403051]. Acknowledgements The authors greatly appreciate the help we received from all of our colleagues and collaborators in these experiments. Conflict of interest: None declared. References 1 Thorgersen EB, Macagno A, Rossetti C, Mollnes TE. Cyanobacterial LPS antagonist (CyP)-a novel and efficient inhibitor of Escherichia coli LPS-induced cytokine response in the pig. Mol Immunol  2008; 45: 3553– 7. Google Scholar CrossRef Search ADS PubMed  2 Mackman N. Lipopolysaccharide induction of gene expression in human monocytic cells. Immunol Res  2000; 21: 247– 51. http://dx.doi.org/10.1385/IR:21:2-3:247 Google Scholar CrossRef Search ADS PubMed  3 Dauphinee SM, Karsan A. Lipopolysaccharide signaling in endothelial cells. 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QJM: An International Journal of MedicineOxford University Press

Published: Mar 1, 2018

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